Uses of THANK, a TNF homologue that activates apoptosis

ABSTRACT

The present invention is directed to the applications of a novel cytokine, named THANK, for TNF homologue that activates apoptosis, NF-κB and c-jun N-terminal kinase. Such applications include using THANK inhibitors to inhibit the activation of NF-κB and to treat a pathological condition caused by the activation of NF-κB. Also provided is a method of inhibiting growth of a wide variety of tumor cells by administering THANK protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 09/496,118, filed Feb.1, 2000 now U.S. Pat. No. 6,475,986, which in turn claims the benefit ofprovisional U.S. Ser. No. 60/118,531, filed Feb. 2, 1999, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of biochemistryand molecular oncology. More specifically, the present invention relatesto uses of a novel cytokine, THANK, a TNF homologue that activatesapoptosis, nuclear Factor-κB, and c-jun N-terminal kinase.

2. Description of the Related Art

In 1984, two homologous cytokines were reported to inhibit the growth oftumor cells specifically (1-7) and was named TNF-α and TNF-β (alsocalled lymphotoxin). Since then over 15 members of this family have beenidentified, including FasL, CD29L, CD30L, CD40L, OX-40L, 4-1BBL, LT-β,TWEAK, TRAIL, RANKL/TRANCE, LIGHT, VEGI, and APRIL (8-16). At the aminoacid sequence level, various members of the TNF family are 20-25%homologous to each other. Most members of this family play an importantrole in gene activation, proliferation, differentiation, and apoptosis.These ligands interact with the corresponding receptor, also members ofthe TNF receptor family, and activate the transcription factors NF-κBand AP1 (9, 17), a stress-activated protein kinase (c-jun N-terminalprotein kinase, JNK), and a cascade of caspases.

The prior art is deficient in the lack of uses of a novel member of theTNF family, named THANK, for TNF homologue that activates apoptosis,NF-κB, and JNK. For example, the prior art is deficient in the lack ofapplications of THANK in inhibiting tumor growth and applications ofTHANK inhibitors in inhibiting the activation of NF-κB. The presentinvention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

By searching an expressed sequence tag (EST) data base using the aminoacid sequence motif of TNF, a novel member of the TNF family, namedTHANK, was identified for TNF homologue that activates apoptosis, NF-κB,and JNK. THANK was primarily expressed by hematopoietic cells. Therecombinant THANK activated NF-κB, c-jun N-terminal kinase, caspase-3and displayed anti-proliferative effects in U937 cells through bindingsites distinct from those for TNF.

The present invention is directed to the applications of THANK,including using THANK inhibitors to inhibit the activation of NF-κB andto treat a pathological condition caused by the activation of NF-κB.Also provided is a method of inhibiting growth of a wide variety oftumor cells by administering THANK protein.

In one embodiment of the present invention, there is provided a methodof inhibiting the activation of NF-κB in cells by treating the cellswith a THANK inhibitor.

In another embodiment of the present invention, there is provided amethod of treating a pathological condition caused by the activation ofNF-κB in an individual by administering a THANK inhibitor in atherapeutically effective amount. Preferably, the pathological conditionis selected from the group consisting of toxic shock, septic shock,acute phase response, viral infection, radiation susceptibility,atherosclerosis, cancer, acute inflammatory conditions, arthritis,allergy, and graft vs. host reaction.

In still another embodiment of the present invention, there is provideda method of inhibiting growth of tumor cells by administering atherapeutically effective amount of THANK protein. Preferably, the cellsare selected from the group consisting of myeloid cells, colon cancercells, prostate cancer cells, breast carcinoma cells, cervical carcinomacells, chronic myeloid leukemic cells and acute myeloid leukemic cells.Still preferably, THANK protein is administered in a dose of from about0.01 mg/kg of patient weight per day to about 100 mg/kg of patientweight per day.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

FIG. 1 shows the full length amino acid sequence of THANK (SEQ ID No.1).

FIGS. 2A and 2B show the amino acid sequence of THANK intracellulardomain (SEQ ID No. 2), transmembrane domain (SEQ ID No. 3),extracellular domain (aa 78-111, SEQ ID No. 4) and the comparison ofTHANK extracellular domain (aa 112-285, SEQ ID No. 5) with mature formof TNF, LT, FasL and LIGHT (SEQ ID Nos. 6-9). Shaded areas indicatehomology with LT, TNF, FasL and LIGHT. FIG. 2C shows SDS-PAGE analysisof THANK (fraction B). FIG. 2D shows western blot analysis of THANK(fraction B). FIG. 2E shows tissue distribution of THANK mRNA. FIG. 2Fshows the expression of THANK mRNA by various cell lines. PBL,perepheral blood leucocytes.

FIG. 3A shows the dose response of THANK-induced NF-κB activation. U937cells (2×10⁶/ml) were treated with different concentrations of THANK for60 min at 37° C. and then assayed for NF-κB by EMSA. FIG. 3B showskinetics of NF-κB activation. U937 cells (2×10⁶/ml) were treated with 1nM of THANK for various lengths of time. FIG. 3C shows supershift andspecificity of NF-κB. Nuclear extract of THANK treated cells (lane 4)were incubated at room temperature for 60 min with anti-p50 (lane 5),anti-p65 (lane 6), mixture of anti-p50 and anti-p65 (lane 7), anti-c-Rel(lane 8), anti-cyclin D1 (lane 9), preimmune serum (lane 10), unlabeledNF-κB oligo nucleotide (lane 2) and then assayed for NF-κB. Lane 1 showsresults for free probe, and lanes 3 and 4 show the THANK-untreated andtreated cells, respectively. FIG. 3D shows effect of anti-THANKpolyclonal antibodies on THANK-induced NF-κB activation in U937 cells.THANK was preincubated with anti-THANK antibodies at a dilution of 1:100or 1:1000 before cells were exposed. FIG. 3E shows effect oftrypsinization and heat denaturation on the ability of THANK to activateNF-κB in U937 cells. THANK was treated with 0.25% trypsin at 37° C. for60 min and then checked for its ability to activate NF-κB (lane 3). Theeffect of trypsin alone is shown in lane 4. THANK was boiled at 100° C.for 10 min, and used for the activation of NF-κB (lane 5). Lane 1 andlane 2 represent NF-κB activation for untreated and THANK treated U937cells, respectively.

FIG. 4A shows the dose response of THANK-induced JNK activation. U937cells (2×10⁶/ml) were treated with different concentrations of THANK for1 h at 37° C. and assayed for JNK activation as described in themethods. Lower panel shows equal loading of protein. FIG. 4B showskinetics of THANK-induced activation of JNK. U937 cells (2×10⁶/ml) weretreated with 1 nM THANK for indicated time period and assayed for JNKactivation. Lower panel shows equal loading of protein.

FIG. 5A shows the dose-dependent cytotoxic effects of THANK against U937cells. 5×10³ cells/well were incubated in triplicate with variousconcentrations of THANK and then examined for cell viability after 72hours. Untreated control is expressed as 100%. FIG. 5B showsTHANK-induced cleavage of PARP in U937 cells. U937 cells (2×10⁶cells/ml) were treated with 0.1, 1 and 10 nM THANK in presence ofcycloheximide (10 μg/ml) for 2 hours at 37° C. In order to compare thecleavage, TNF was used as a positive control. FIG. 5C shows competitiveinhibition of labeled TNF binding to U937 cells by unlabeled TNF (20 nM)and THANK (150 nM). U937 cells (0.5×10⁶ cells/well) were incubated with0.25×10⁶ cpm of ¹²⁵I-TNF in ice bath for 1 hour in presence or absenceof the unlabeled competitors. Cell-bound radioactivity was measured in agamma counter. Results are expressed as mean±S.D.

DETAILED DESCRIPTION OF THE INVENTION

Using the amino acid sequence motif of TNF, an EST database wassearched. A novel full-length cDNA encoding 285 amino acid residues (SEQID No. 1, FIG. 1) was identified, and named THANK. THANK is a type IItransmembrane protein with 15-20% overall amino acid sequence homologyto TNF, LT-α, FasL and LIGHT, all members of the TNF family. The mRNAfor THANK was expressed at high levels by peripheral blood leukocytes,lymph node, spleen, and thymus and at low levels by small intestine,pancreas, placenta, and lungs. THANK was also prominently expressed inhematopoietic cell lines. The recombinant purified protein expressed inthe baculovirus system had an approximate molecular size 20 kDa withamino terminal sequence of LKIFEPP (SEQ ID No. 10). Treatment of humanmyeloid U-937 cells with purified THANK activated NF-κB consisting ofp50 and p65. Activation was time- and dose-dependent, beginning with aslittle as 1 pM of the cytokines and as early as 15 min. Under the sameconditions, THANK also activated c-jun N-terminal kinase (JNK) in U937cells. THANK also strongly suppressed the growth of tumor cell lines andactivated caspase-3. Although THANK had all the activities and potencyof TNF, it did not bind to the TNF receptors, which indicates that THANKis a novel cytokine that belongs to the TNF family and activatesapoptosis, NF-κB, and JNK through a distinct receptor.

The present invention is directed to various applications of THANK,including using THANK inhibitors to inhibit the activation of NF-κB andto treat a pathological condition caused by the activation of NF-κB.Also provided is a method of inhibiting growth of a wide variety oftumor cells by administering THANK protein.

In one embodiment of the present invention, there is provided a methodof inhibiting the activation of NF-κB in cells by treating the cellswith a THANK inhibitor.

In another embodiment of the present invention, there is provided amethod of treating a pathological condition caused by the activation ofNF-κB in an individual by administering a THANK inhibitor in atherapeutically effective amount. Preferably, the pathological conditionis selected from the group consisting of toxic shock, septic shock,acute phase response, viral infection, radiation susceptibility,atherosclerosis, cancer, acute inflammatory conditions, arthritis,allergy, and graft vs. host reaction.

In still another embodiment of the present invention, there is provideda method of inhibiting growth of tumor cells by administering atherapeutically effective amount of the THANK protein. Preferably, theTHANK protein is used to treat tumor cells such as myeloid cells, coloncancer cells, prostate cancer cells, breast carcinoma cells, cervicalcarcinoma cells, chronic myeloid leukemic cells and acute myeloidleukemic cells. Generally, the THANK protein may be adminstered in anypharmacological dose which inhibits or kills tumors. Preferably, theTHANK protein is administered in a dose of from about 0.01 mg/kg ofpatient weight per day to about 100 mg/kg of patient weight per day.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1

Identification, Cloning, Expression, and Purification of THANK

Using high throughput automated DNA sequence analysis of randomlyselected human cDNA clones, a database containing more than two millionESTs obtained from over 750 different cDNA libraries was been generatedby Human Genome Sciences, Inc. A specific homology and motif searchusing the known amino acid sequence motif of TNF family members againstthis database revealed several ESTs having homology to members of theTNF family. One full length cDNA clone (HNEDU15) encoding an intactN-terminal signal peptide was isolated from a human neutrophil libraryand selected for further investigation. The complete cDNA sequence ofboth strands of this clone was determined, and its homology to TNF wasconfirmed. This gene product was named THANK.

THANK is a 285 amino acid long type II transmembrane protein (SEQ ID No.1, FIG. 1). The intracellular domain was found to be located betweenamino acid residues 1 through 46 (SEQ ID No. 2), and the transmembranedomain between amino acid residues 47 through 77 (SEQ ID No. 3) (FIGS.2A and 2B).

The cDNA encoding the extracellular domain of THANK (aa 78-111, SEQ IDNo. 4 and 112-285, SEQ ID No. 5) was amplified employing the PCRtechnique using the following primers: 5′ BamHI,GCGGGATCCCAGCCTCCGGGCAGAGC (SEQ ID No. 11) and 3′ XbaI,GCGTCTAGATCACAGCACTTTCAATGC (SEQ ID No. 12). The amplified fragment waspurified, digested with BamHI and XbaI, and cloned into a baculovirusexpression vector pA2-GP, derived from pVL94. The cloning, expressionand confirmation of the identity of the cloned product were performedusing standard techniques (18).

Recombinant THANK was purified from the clarified culture supernatant of92 h post-infected Sf9 cells. The protein was stepwise purified bycation and anion exchange chromatography. The purified THANK wasanalyzed for purity by 12% SDS-PAGE and by western blot analysis.

EXAMPLE 2

Northern Blot Analysis

Two multiple human tissue northern blots containing 2 μg of poly (A)⁺RNA per lane of various tissues (Clontech, Palo Alto, Calif.) wereprobed with ³²P-labeled THANK cDNA. RNA from a selected panel of humancell lines were probed following the same technique.

EXAMPLE 3

Production of THANK Antibodies

Antibodies against THANK were raised by injecting 0.2 mg purifiedrecombinant antigen in Freund's complete adjuvant (Difco Laboratories)subcutaneously into a rabbit. After three weeks, the injection wasrepeated and the rabbit was bled every third week. The specificity ofthe antiserum was tested by ELISA and western blot.

EXAMPLE 4

Receptor-Binding Assay

TNF receptor-binding assay was performed following a modified procedurepreviously described (19). Briefly, 0.5×10⁶ cells/well (triplicate well)in 100 μl binding medium (RPMI-1640 containing 10% FBS) were incubatedwith ¹²⁵I-labelled TNF (2.5×10⁵ cpm/well, specific activity 40 mCi/mg)either alone (total binding) or in the presence of 20 nM unlabeled TNF(nonspecific binding) or 150 nM unlabeled THANK in an ice bath for 1 h.Thereafter, cells were washed three times with ice-cold PBS containing0.1% BSA to remove unbound ¹²⁵I-TNF. The cells were dried at 80° C., andthe cell bound radioactivity was determined in a gamma counter(Cobra-Auto Gamma, Packard Instrument Co.)

EXAMPLE 5

Electrophoretic Mobility Shift Assay (EMSA)

NF-κB activation was analyzed by EMSA as described previously (20, 21).In brief, 6 μg nuclear extracts prepared from THANK-treated cells wereincubated with ³²P-end-labeled 45-mer double-stranded NF-κBoligonucleotide for 15 min at 37° C., and the DNA-protein complexresolved in 7.5% native polyacrylamide gel. The specificity of bindingwas examined by competition with unlabeled 100-fold excessoligonucleotide. The specificity of binding was also determined bysupershift of the DNA-protein complex using specific and irrelevantantibodies. The samples of supershift experiments were resolved on 5.5%native gels. The radioactive bands from dried gels were visualized andquantitated by PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.)using ImageQuant software.

EXAMPLE 6

Western Blot of THANK

Purified THANK sample was resolved on 12% SDS-PAGE, electrotransferredto a nitrocellulose membrane, and probed with polyclonal antibodies(1:6000) raised in rabbits. The blot was then treated withHRP-conjugated secondary antibodies and finally detected bychemiluminescence (ECL, Amersham Pharmacia Biotech. Arlington Heights,Ill.).

EXAMPLE 7

c-Jun Kinase Assay

The c-Jun kinase assay was performed by a modified method as describedearlier (22). Briefly, 100-μg cytoplasmic extracts were treated withanti-JNK1 antibodies, the immunocomplexes were precipitated with proteinA/G-Sepharose beads (Pierce, Rockford, Ill.) and assayed for theenzymatic activity by using glutathione S-transferase-Jun (aa 1-79) assubstrate (2 μg) in the presence of 10 μCi [³²P]ATP. The kinase reactionwas carried out by incubating the above mixture at 30° C. in kinaseassay buffer for 15 minutes. The reaction was stopped by adding SDSsample buffer, followed by boiling. Finally, protein was resolved on a9% acrylamide gel under reduced conditions. The radioactive bands of thedried gel were visualized and quantitated by phosphorImager as mentionedpreviously.

To determine the total amount of JNK1 protein, 30 μg of the cytoplasmicextracts were loaded on 9% acrylamide gels. After electrophoresis, theprotein was transferred to nitrocellulose membranes, blocked with 5%non-fat milk protein and probed with rabbit polyclonal antibodies(1:3000) against JNK1. The blots were then reacted with HRP-conjugatedsecondary antibodies and finally detected by chemiluminescence (ECL,Amersham)

EXAMPLE 8

Cytotoxicity Assays

The cytotoxic effects of THANK against tumor cells were measured bymodified tetrazolium salt (MTT) assay described earlier (23) and by itsability to activate caspase-3 leading to cleavage of poly (ADP-ribose)polymerase (PARP) (24). For cytotoxicity, 5×10³ cells in 0.1 ml wereplated in triplicate in 96-well plates and exposed to variableconcentrations of either THANK or TNF (for comparison) in 0.1 ml. After72 h incubation at 37° C., cells were examined for viability. Toestimate caspase-3 activation by PARP cleavage, cell extracts (50μg/sample) were resolved on 7.5% acrylamide gels, electrophoresed,transferred to nitrocellulose membranes, blocked with 5% non-fat milkprotein, probed with PARP monoclonal antibody (1:3000) and detected byECL as indicated above.

EXAMPLE 9

Identification, Sequence, and Purification of THANK

The predicted amino acid sequence of mature THANK (112-285, SEQ ID No.5) is 15%, 16%, 18% and 19% identical to LIGHT, FasL, TNF arid LT-α,respectively (FIGS. 2A and 2B). The cDNA for this novel cytokine wascloned and expressed in a baculovirus expression system. In CM cellulosecation-exchange chromatography, THANK eluted first with 1 M NaCl(fraction A) and then with 1.5 M NaCl (fraction B). Fractions A and Bhad approximate molecular mass of 23 kDa and 20 kDa, respectively on 12%SDS-PAGE (FIG. 2C); and amino terminal sequences of LKIFEPP (SEQ ID No.10) and AVQGP (SEQ ID No. 13) starting at AA112 and AA134, respectively.An apparently higher molecular size obtained by SDS-PAGE than thatpredicted from the number of amino acids suggested a post-translationalmodification. The amino acid sequence of the mature THANK lacked,however, the potential N-glycosylatio site. Polyclonal antibodiesprepared against THANK recognized the cytokine on western blot (FIG.2D).

EXAMPLE 10

Tissue and Cell Line Distribution of THANK

Northern blot analysis indicated that THANK was expressed in peripheralblood leukocytes (PBL), spleen, thymus, lung, placenta, small intestineand pancreas; with highest expression in PBL (FIG. 2E). Analysis of thecell line blot (Clonetech Inc.) revealed very high expression in HL60,detectable expression in K562, A549, and G361, and no detectabletranscript in HeLa, MOLT4, Raji, and SW480 cell lines. Thus cells andtissues of the immune system expressed THANK transcripts.

EXAMPLE 11

THANK Activates NF-κB

One of the earliest events activated by most members of the TNFsuperfamily is NF-κB activation (25). The results depicted in FIGS. 3A &3B indicate that THANK activated NF-κB in a dose and time-dependentmanner. Less that 10 pM THANK was enough to activate NF-κB in U937cells, though peak activation was obtained at 1 nM (FIG. 3A). THANKinduced optimum NF-κB activation within 60 min at 1 nM; no significantincrease was thereafter (FIG. 3B). The gel shift band was specific, asits formation could be eliminated with excess unlabeled oligonucleotide.It was supershifted by anti-p50 and anti-p65 antibodies only (FIG. 3C),thus indicating that the nuclear factor was composed of p50 and p⁶⁵subunits. No significant difference was found in the ability to activateNF-κB between 20 and 23 kDa forms of THANK indicating that residues 112through 134 are optional for the biological activity (data not shown).

To ascertain that the observed activation was due to THANK and not acontaminant, the protein was preincubated with anti-THANK polyclonalantibodies before treatment with the cells. FIG. 3D shows a lack ofNF-κB activation after treatment of THANK with antibodies even at a 1 to1000 dilution. Antibody against THANK by itself had no effect. Tofurther ascertain that NF-κB activation was due to the proteinaceousnature of THANK, the protein was either digested with trypsin orheat-denatured prior to treatment. Both treatments completely abolishedNF-κB activation in U937 cells, confirming that THANK was responsiblefor this activation (FIG. 3E). Although THANK was as potent as TNF withrespect to both dose and time required for NF-κB activation, the overallamplitude of response was less with THANK. In this respect the activityof THANK was comparable with LT-α (21).

EXAMPLE 12

THANK Activates c-Jun N-terminal Kinase

The activation of c-Jun kinase (JNK) is another early event that isinitiated by different members of the TNF family (17, 22). THANKactivated JNK activity in a time- and dose-dependent manner (FIGS. 4A &4B). At 10 pM the activity increased by 2.5-fold; at 1 nM it reached 4.4fold. An additional increase in THANK concentration slightly decreasedactivation (FIG. 4A). The peak activation of JNK was observed at 60 min(3.3-fold increase), which gradually decreased thereafter (FIG. 4B).These results suggest that, like TNF, THANK transiently activates JNK inU937 cells. The activation of JNK by THANK was not due to an increase inJNK protein levels, as immunoblot analysis demonstrated comparable JNK1expression at all dose and time points (FIGS. 4A & 4B, lower panels)

EXAMPLE 13

THANK-Induced Cytotoxicity and Caspase-3 Activation

Activations of NF-κB and JNK are early cellular responses to TNF, whichare followed by cytotoxic effects to tumor cells. The effect ofdifferent concentrations of THANK on the cytotoxic effects against tumorcell lines was examined and compared with that of TNF.

Results in FIG. 5A show that THANK inhibited the growth of humanhistiocytic lymphoma U-937 cells in a dose-dependent manner. BesidesU-937 cells, THANK also inhibited the growth of prostate cancer (PC-3)cells, colon cancer cells (HT-29), cervical carcinoma cells (HeLa),breast carcinoma cells (MCF-7), and embryonic kidney cells (A293) (datanot shown). The growth inhibition curve of THANK was superimposable withthat of TNF, indicating comparable potency.

Degradation of PARP by caspase-3 is one of the hallmarks of apoptosis intumor cells (26). It was found that treatment of U-937 cells with THANKfor 2 h induced partial cleavage of PARP in U937 cells, whereas TNFalmost completely cleaved PARP under these conditions (FIG. 5B). Thissuggests that THANK can activate caspase-3, though not so strongly asTNF.

EXAMPLE 14

THANK Binds to Receptors Distinct from TNF Receptors

It was previously shown that TNF and LT, which share homology with eachother to the same extent as THANK, bind to the same cell surfacereceptors (4). Since THANK has significant amino acid sequence homologywith TNF, and like TNF exhibits cytotoxic effects, and activates NF-κBand JNK, its binding to the TNF receptor was examined. The receptorbinding results (FIG. 5C) show that 20 nM unlabeled TNF almostcompletely blocked the binding of ¹²⁵I-labeled TNF to U-937 cells,whereas 150 nM unlabeled THANK did not compete for ¹²⁵I-TNF bindingsites. These results suggest that THANK interacts with U937 cellsthrough a receptor distinct from that for TNF.

In summary, a novel cytokine expressed by hematopoietic cells wasidentified, which can, like TNF and LT-α, activate NF-κB and JNK andinhibit the growth of a wide variety of tumor cells. Although thestructure of THANK also exhibits homology to FasL and LIGHT, the latterhave not been reported to activate NF-κB. Preliminary results by usingflow cytometry indicate that THANK protein is expressed bypromyelomonocytic HL-60 cells (data not shown). Because THANK isexpressed by hematopoietic cells, it appears to be similar to LT-α anddissimilar from other members of the TNF superfamily. Among all themembers of the TNF superfamily, THANK exhibits cytotoxic effects similarto TNF and LT-α. Whether THANK exhibits immunomodulatory activities andin vivo antitumor activities is currently under investigation.

The following references were cited herewith.

-   1. Aggarwal, et al., 1984 J Biol Chem, 259:686-691.-   2. Gray, et al., 1984 Nature, 312:721-724.-   3. Pennica, et al., 1984 Nature, 312:724-729.-   4. Aggarwal, et al., 1985 Nature, 318:665-667.-   5. Aggarwal, et al., 1985 J Biol Chem, 260:2334-2344.-   6. Aggarwal, et al., J Biol Chem, 260:2345-2354.-   7. Sugarman, et al., 1985 Science, 230:943-945.-   8. Aggarwal, et al., 1996 Eur. Cytokine Netw. 7: 93-124.-   9. Smith, et al., 1994 Cell 76: 959-962.-   10. Wiley, et al., 1995 Immunity. 8:21-30.-   12. Hahne, et al., 1998. J. Exp. Med. 188:1185-90.-   13. Chicheportiche, et al., J. Biol. Chem. 272: 32401-32410.-   14. Zhai, et al., FASEB J. (In press).-   15. Anderson, et al., Nature. 390:175-9.-   16. Wong, et al., J. Biol. Chem. 272: 25190-4.-   17. Singh, et al., 1998 J. Interferon and Cytokine Res. 18, 439-450.-   18. Ni, et al., 1997 J. Biol. Chem. 272: 10853-10858.-   19. Higuchi, et al., 1992 Anal. Biochem. 204: 53-57.-   20. Schreiber, et al., 1989 Nucleic Acids Res. 17: 6419-6422.-   21. Chaturvedi, et al., 1994 J. Biol. Chem. 269: 14575-14583.-   22. Kumar, et al., 1998. Methods in Enzymology, Vol. 000 (ed. L.    Packer), Academic Press, pp. 339-345.-   23. Hansen, et al., 1989 J. Immunol. Methods. 119: 203-210.-   24. Haridas, et al., 1998 J. Immunol. 160, 3152-3162.-   25. Baeuerle, et al., 1996 Cell 87:13-20.-   26. Tewari, et al., 1995. Cell 81:801-9.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1. A method of inhibiting the activation of nuclear factor-κB in cancercells to thereby induce radiation susceptibility therein, comprising thesteps of treating said cells with an inhibitor of THANK protein in anamount effective to induce radiation sensitivity therein, wherein theTHANK inhibitor is an antibody that recognizes THANK protein, andwherein said THANK protein is defined by the amino acid sequence of SEQID NO:1.